Hyperadhesive von Willebrand Factor Promotes Extracellular Vesicle-Induced Angiogenesis

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SUMMARY
Bleeding associated with left ventricular assist device (LVAD) implantation has been attributed to the loss of large von Willebrand factor (VWF) multimers to excessive cleavage by ADAMTS-13, but this mechanism is not fully supported by the current evidence. We analyzed VWF reactivity in longitudinal samples from LVAD patients and studied normal VWF and platelets exposed to high shear stress to show that VWF became hyperadhesive in LVAD patients to induce platelet microvesiculation. Platelet microvesicles activated endothelial cells, induced vascular permeability, and promoted angiogenesis in a VWF-dependent manner. Our findings suggest that LVAD-driven high shear stress primarily activates VWF, rather than inducing cleavage in the (GI) bleeding is most common and is found at the site of angiodysplasia in more than 50% of cases. 12,13 Post-LVAD GI bleeding has been shown to be reduced in patients receiving angiotensin-converting enzyme inhibitors or angiotensin receptor blockage after LVAD implantation, 14,15 suggesting that angiogenesis is a causal factor for LVAD-induced bleeding.
Nearly all LVAD patients lose large VWF multimers in a condition called acquired von Willebrand syndrome (aVWS). aVWS develops soon after LVAD implantation, resolves rapidly after LVAD explantation, and is not observed in heart transplant recipients, 16,17 suggesting that it is caused by hydrodynamic changes in the LVAD-driven blood flow. where VWF is enriched in ultra-large multimers (ULVWF) that are intrinsically hyperactive in binding platelets and endothelial cells. 18,19 Upon release, these ULVWF multimers are anchored to the endothelium and rapidly but partially cleaved by ADAMTS-13. [19][20][21][22] The cleavage converts constitutively hyperadhesive ULVWF multimers 23,24 into plasma VWF multimers that bind circulating platelets poorly but can be activated to increase their adhesive activity by high shear stress (HSS). 23,24 HSS at similar levels has been shown to reduce VWF adhesive activity by exposing the cryptic  Hyperadhesive VWF-Induced Aberrant Angiogenesis M A R C H 2 0 2 2 : 2 4 7 -2 6 1 ADAMTS-13, 26 but the role of shear-induced VWF activation is not known.
We investigated VWF cleavage and activation under LVAD-driven circulation by: 1) studying longitudinal samples from LVAD patients; 2) identifying shear-induced structural changes of VWF in vivo and in vitro; and 3) dissecting the interplay between extracellular vesicles (EVs) and hyperadhesive VWF in promoting the aberrant angiogenesis that could be the cause of the angiodysplasia found in the mucosal tissue of LVAD patients.

RESULTS
Among the 26 patients (Table 1) The levels of total VWF-bound EVs (VWF þ /PS þ ) were significantly higher in patients at baseline than in the control subjects ( Figure 1E). They were reduced moderately post-LVAD but increased again at comparable levels between patients with bleeding and those with thrombosis. In contrast, CD41 þ /VWF þ EVs from VWF-activated platelets (pEVs) were significantly increased after LVAD implants and increased further at clinical events ( Figure  collected at bleeding or thrombotic events than on those collected outside of these events ( Figure 1G).
Together, these data suggest that EVs in plasma from patients at the baseline activated ECs to increase permeability and that this activity increased in patients with bleeding and was associated with pEVs, which were significantly increased in post-LVAD samples.
The results reported in Figure 1 led us to hypothesize that VWF þ pEVs carry angiogenic activity because increased permeability is considered to be an early stage of angiogenesis. When plasma was fractionated, we found that EVFP ( To specifically examine the effects of pEVs, we  Figure 3C), which were reduced by the GP Iba antibody AK2. This antibody blocks VWF binding to GP Iba on platelets. 29 We also detected sheardependent hemolysis (Supplemental Figure 3). The pEVs from platelets exposed to HSS promoted angiogenesis in a VWF-dependent manner, but those from unsheared platelet-rich plasma did not ( Figures 3D to 3F). The vascular sprouts from AVSs cultured in pEV-supplemented GPM were shorter, denser, and hairlike ( Figure 3E), similar to those induced by EVs from LVAD patients ( Figure 2). These pEVs contained 537.7 AE 36.6 pg/mL of VEGF (n ¼ 6), significantly higher than that in the heterogeneous EVs from LVAD patients (n ¼ 16; t test, P ¼ 0.011).
The CD31 þ microvessels from AVSs cultured in pEVsupplemented GPM were small and often lacked intact vascular lumens ( Figure 3I), whereas those in GRM were fully developed ( Figure 3J). The number of CD31 þ vessels was higher in AVSs cultured with pEV and was reduced by the VWF antibody ( Figure 3K).
For validation, we also used the 2-dimensional model of endothelial network formation assay to investigate the synergistic effects of pEVs and VWF on angiogenesis. Consistent with the data in Figure 3,  Figure 4G). We also measured VWF cleaved by ADAMTS-13 and uncleaved VWF in EV and EV-free fractions of plasma samples collected at discharge of LVAD patients using mass spectrometry. 30 The ratio of cleaved VWF in EVFP to that in EV fraction was 2.27 after adjustment for total VWF, suggesting that platelet-and pEV-bound VWF was significantly less cleaved. Together, these results suggest that: 1) the ECs underwent persistent exocytosis of VWF after LVAD implants and were subjected to additional stress at the time of severe bleeding or thrombosis; 2) plasma VWF in LVAD patients was   techniques. First, the co-immunoprecipitation assay showed that a recombinant A2 protein bound to the exposed A1 domain 28 to form a complex with VWF in the plasma samples of LVAD patients ( Figure 5C). In contrast, A2 bound VWF from healthy subjects only in the presence of ristocetin ( Figure 5C), which activates VWF to bind its platelet receptor. 29 Second, A2 blocked SIPA ( Figure 5D), which is induced by the binding of shear stress-activated VWF to platelets. 25 Third, the thiol-containing VWF accounted for 86.4% AE 7.1% in healthy subjects but was reduced to 61.0% AE 8.1% in patients at baseline and decreased further to 42.3% AE 13.0% after LVAD implantation ( Figure 5E), with a parallel increase of VWF in the supernatant (Supplemental Figure 6). These results suggest that VWF multimers in LVAD patients were oxidized and underwent conformational changes to expose the A1 domain.
Exposing normal platelet-rich plasma to HSS for 5 minutes failed to induce VWF cleavage ( Figure 5F), but it did induce significant platelet activation and aggregation by VWF (Figures 3A to 3C). As a control, VWF was cleaved in static conditions after incubation for 16 hours in the presence of 1.5 M urea and 1 mM of BaCl 2 . An isolated A2 required 1 hour to be cleaved without added chemicals ( Figure 5G). In contrast, VWF was partially cleaved after exposure to HSS for 60 minutes ( Figure 5H). The cleavage was not affected by platelets (2 Â 10 5 /mL) or erythrocytes (2 Â 10 6 /mL), but it was prevented by collagen (10 mg/mL). The cleavage was similarly induced under a turbulent flow generated in a vortex   Figure 6A); and 2) we have shown that VWF maintains its shear-induced active conformation for more than 5 hours after shear exposure has stopped, 32 allowing sufficient time for experiments to be conducted. Hemostasis was restored partially in VWF -/mice infused with VWF and completely with the VWF that was exposed to HSS for 5 minutes at 37 C ( Figure 6B, Supplemental Figure 8). Platelets from VWF -/mice infused with sheared VWF expressed CD62p ( Figure 6C), developed moderate thrombocytopenia ( Figure 6D) and generated more VWF þ pEVs ( Figure 6E). These mice also had elevated levels of endothelial EVs ( Figure 6F). These results suggest that VWF exposed to HSS fully restored the hemostasis of VWF -/mice.  and 120 dynes/cm 2 and **P < 0.01 vs HS. Abbreviations as in Figure 1.
VWF before and after LVAD implantation. We made several novel observations.
First, the EVs from LVAD patients induced significant vascular permeability and aberrant angiogenesis in a VWF-dependent fashion (Figures 1 and 2).
This finding is consistent with previous reports that EVs can transmigrate through the endothelial barrier 38,39 and that VWF plays a role in the process. 28,40 VWF did not promote angiogenesis directly, but likely served as a coupling factor that tethered pEVs to ECs in flowing blood, likely through simultaneous binding to GP Iba on pEVs and a v b 3 integrin and CD62p on ECs 41,42 to locally concentrate VEGF for angiogenesis. 43 VEGF is stored in the a-granules of platelets, which releases it upon activation. 44 VEGF induces VWF release from ECs, 45 causes endothelial permeability, and promotes the formation of immature and "leaky" vessels. 46 VEGF is also carried by EVs in patients with diabetes mellitus. 47 In addition, EVs from endothelial cells contain angiopoietin-like protein 2, 48 which also promotes angiogenesis. Our finding is supported by previous reports that EVs deriving from adipocytes and leukocytes promote angiogenesis 49,50 through distinct but closely related pathways. [50][51][52][53] It is also interesting to note that EVderived proangiogenic activity was enhanced in a swine model of obesity and hyperlipidemia, 54 which are key causal factors for the coronary heart disease that could lead to heart failure. EVs can also carry molecules that may be inhibitory to angiogenesis, such as miR-24, 55 which inhibits cell proliferation.
Second, we show that the VWF in LVAD patients was hyperadhesive, activating platelets ( Figure 1) and enhancing VWF:CB under static and flow conditions ( Figure 4). VWF:CB has been previously reported to both increase 56,57 and decrease in LVAD patients. 35,58,59 However, reports on the latter compare VWF:CB before and after LVAD. This comparison may obscure the true level of VWF adhesive activity because patients with end-stage heart failure have elevated levels of VWF in comparison with healthy subjects, [60][61][62] leading to the impression that VWF:CB is reduced in LVAD patients. We found that VWF:CB was moderately reduced post-LVAD, but it remained higher than that of healthy subjects.
The finding that VWF lost large multimers but remained hyperadhesive, albeit at reduced levels ( Figure 4), defines a GOF phenotype with large VWF multimers lost to enhanced binding to platelets, consistent with increased VWF on platelets and pEVs found in the post-LVAD samples ( Figure 1). Consistent with the notion, VWF found on pEVs was significantly less cleaved than VWF found in plasma, suggesting that platelets were activated to generate pEVs by less cleaved and thus more adhesive VWF, which also mediated EV-EC interaction to promote angiogenesis.
This GOF phenotype is supported by the finding that the VWF exposed to HSS was more effective in restoring hemostasis in VWF-null mice ( Figure 6),    65 We show that the A1 and A2 complexes are disassociated to expose the A1 domain of VWF in LVAD patients ( Figure 5C), as schematically illustrated in Figure 7B, but did not induce cleavage likely because the VWF was oxidized ( Figure 5E   A2 complex so that the A1 exposed (activated) VWF can form a complex with isolated A2. (C) Cysteine thiols of VWF can be oxidized to form intermultimeric disulfide bonds under HSS and thus cannot be precipitated by the active thiol beads, which form mix disulfide bonds with surface exposed free thiols on VWF. 68 Abbreviations as in Figures 1 and 3. compare VWF profiles so that we will be able to more precisely define the impact of shear stress on VWF.
Third, because of a limited plasma volume, we were unable to map the specific amino acids involved in forming intermultimer disulfide bonds or being oxidized. We are developing a new mass spectrometric protocol to overcome this technical difficulty.
Finally, because of the lack of suitable research on LVADs in mouse models, we were unable to test our hypothesis of LVAD-induced GOF VWF and its synergistic actions with pEVs to promote angiogenesis in vivo. We can overcome this obstacle in experiments on large animals in the future.

CONCLUSIONS
In summary, we demonstrate that VWF was not excessively cleaved but became hyperadhesive in the majority of LVAD patients. We identified platelet microvesiculation, resulting from the LVAD-driven HSS blood flow, as a key contributor to the hemostatic dysfunction, endotheliopathy, and aberrant angiogenesis found in LVAD patients. These findings could have a direct impact on the clinical management of patients on LVAD support. For example, aspirin, which is commonly prescribed after LVAD, may not be effective in preventing this VWFmediated platelet activation and EV-driven angiogenesis because shear-induced platelet activation is insensitive to aspirin. 70,71 Another finding that requires further investigation is that all patients received dipyridamole post-LVAD, which is a phosphodiesterase inhibitor that reduces platelet reactivity and vascular tension by blocking the adenosine metabolism by erythrocytes and vascular endothelial cells. A recent study found that the phosphodiesterase 3A inhibitor reduced platelet microvesiculation, 72 but dipyridamole failed to reduce plasma levels of pEVs in the present study. These findings are also significant for understanding the complications associated with other medical devices that significantly alter blood hydrodynamics (eg, percutaneous microaxial LVAD and extracorporeal membrane oxygenation).

FUNDING SUPPORT AND AUTHOR DISCLOSURES
The authors have reported that they have no relationships relevant to the contents of this paper to disclose. These diverse activities derive from the size and structure of VWF multimers. How hydrodynamic changes induced by LVAD-driven blood flow alter VWF structure remains poorly understood. This study shows that VWF is predominantly activated by HSS and oxidative stress, calling for more comprehensive study of VWF in the pathogenesis of LVAD-induced bleeding complications.

ADDRESS
TRANSLATIONAL OUTLOOK: The current study used extensive technologies to define the structure-and-function correlation of VWF multimers found in LVAD-driven blood flow. The study raises the question as whether VWF multimers are excessively cleaved by the metalloprotease ADAMTS-13 or activated to bind platelets, and more importantly, how the 2 opposing processes reach the equilibrium in patients on LVAD supports.
This study identified shear-induced and VWF-mediated platelet microvesiculation as a potential key contributor to angiodysplasia and associated bleeding in patients on LVAD supports. The clinical and laboratory VWF variables obtained from this study could allow us to develop a composite score that predicts the bleeding risk and need for targeted prophylaxis of individual patients based on their VWF profiles.